Nitric oxide (NO) is a potent biologic mammalian mediator which has triggered an explosion of scientific studies over the last decade. This research has identified diverse physiologic and pathophysiologic roles for NO as a vasodilator, neurotransmitter, antimicrobial effector molecule, and immunomodulator. Inducible nitric oxide synthase (iNOS) is a gene which is expressed in most tissues in response to combinations of inflammatory cytokines. Therefore, it is important to understand the mechanisms by which the human iNOS gene expression is regulated. We are interested in the synergistic regulation of iNOS gene transcription by combinations of cytokines including TNFalpha, IL-1B, and IFNg. Using deletional and site-specific mutagenesis of human iNOS promoter- luciferase reporter plasmids, we have identified a cytokine-responsive enhancer element located between -5.2 and -6.1 kb of 5'-flanking DNA that contains multiple binding sequences for NF-kB and Stat1. An overlapping NF-kB-Stat1 element located at -5.8 kb appears particularly important for induction of iNOS by cytokines. We propose that the association of Stat1 with p65 NF-kB is important for the synergistic induction of human iNOS transcription by either TNFa or IL-1B, signaling in combination with IFNg. Here, we design an experimental strategy that will test the functional importance of the p65-Stat1 interaction in regulating iNOS transcription. The information gained will increase our understanding of the control of iNOS transcription, describe novel mechanism(s) of cytokine-synergy in signal transduction, and will be important in designing therapeutic strategies against pathophysiologic conditions where cytokine expression is relevant.